Proteinaceous molecules, such as enzymes, hormones, storage proteins, binding proteins and transport proteins may be produced by recombinant DNA techniques. For instance, DNA fragments coding for a selected protein, together with appropriate DNA sequences for a promoter and ribosome binding site are ligated to a plasmid vector. The plasmid is inserted within a host prokaryotic or eukaryotic cell. Transformed host cells are identified, isolated and then cultivated to cause expression of the proteinaceous molecules.
The desired protein is then isolated from the culture medium and purified by a variety of techniques employed either individually or in combination. These purification procedures may include techniques to segregate the desired protein based on its molecular size. Such procedures include dialysis, density-gradient centrifugation and gel column chromatography. Dialysis and density-gradient centrifugation, however, are not selected enough to highly purify protein. While the use of gel column chromatography results in greater purification, many of the desired protein molecules are lost during the purification process, thereby resulting in a low yield.
Protein molecules also may be separated from mixture by procedures based on solubility differences. For instance, isoelectric precipitation takes advantage of the change in solubility of proteins as a function of pH while solvent fractionation employs the fact that the solubility of proteins vary as a function of the dielectric constant of the medium. Neutral salts, such as ammonium sulfate, are used to precipitate out proteins as a result of decreased protein solubility based on the high ionic strength of the salt. A severe drawback of solvent fractionation is that solvents can cause the proteins to denature. Neither isoelectric precipitation nor salt precipitation are able to purify proteins beyond a moderate level. One advantage of salt precipitation, however, is that it typically gives close to a 100% yield, and thus this method is often employed as an initial step in tandem with other procedures.
Proteins also may be separated based on their ionic properties, for instance, by various types of electrophoresis or by ion-exchange chromatography. Most electrophoresis techniques are used as analytical tools and are not practical on a large scale basis. While ion-exchange chromatography can result in highly purified proteins, the yield level is typically very low, with many of the protein molecules either being lost in prior eluates or remaining bound to the column matrix.
Affinity chromatography often is employed to avoid the negative aspects of the above-mentioned purification procedures including ion-exchange chromatography and gel column chromatography. Affinity chromatography is based on the capacity of proteins to bind specifically and noncovalently with a ligand. Used alone, it can isolate proteins from very complex mixtures with not only a greater degree of purification than possible by sequential ion-exchange and gel column chromatography, but also without significant loss of activity. See Rosenberry et al., "Purification of Acetylcholinesterase by Affinity Chromatography and Determination of Active Site Stoichiometry," 247 Journal of Biological Chemistry, 1555-1565 (1972). Although affinity chromatography can produce a high level of protein purification, this technique requires the availability of significant amounts of the corresponding ligand (for instance, antibody for antigen or substrate for enzyme) for the protein molecule being isolated. Thus, it may be necessary to carry out a time-consuming, laborious regime of innoculating mice or other animals with the protein molecule of interest in purified form and then identifying a specific ligand for the protein molecule. Thereafter, the ligand must be amplified, for instance, by hybridoma techniques and then purified for covalent detachment to the affinity column matrix.
It will be appreciated that it may be very difficult to isolate a specific ligand for certain protein molecules. Moreover, specific ligands do not exist for all types of protein molecules, such as certain enzymes. As a consequence, to date, affinity chromatography has not been employed as a universal isolation and purification technique for all protein molecules.
Accordingly, it is a principle object of the present invention to use recombinant DNA techniques for economically producing a desired protein and then efficiently purifying the protein.
It is a specific object of the present invention to provide an affinity purification process wherein a single ligand may be employed to isolate and purify substantially all protein molecules expressed by transformed host cells, whether antigenic or not.
A further particular object of the present invention is to provide a standard, highly efficient process that can be used on a small research level or a large commercial scale to purify substantially all protein molecules produced by recombinant DNA techniques.
An additional particular object of the present invention is to provide technique that is capable of highly purifying substantially any protein molecule generated by recombinant DNA techniques in a single, affinity chromatography step, but without sacrificing high yields.